Project 2 Abstract This project bridges long-acting antiretroviral therapeutic nanoformulation (nanoART) synthesis, viral tissue reservoir targeting and pharmacodynamic tests in rodents and large animal confirmatory studies when required. Three aims joint the project. The first, seeks to determine the effectiveness of the targeted drug formulations for sustained antiretroviral and immune responses in chronically HIV-1 infected humanized mice treated with decorated or targeted nanoART. Reductions in viral load and restored CD4+ T cell numbers will be assessed when compared against results seen with native drug, formulations containing untargeted ART and those animals treated with an irrelevant control drug (for example, fluconazole). Parallel tests for potential tissue and cellular toxicities and ?putative? drug-drug interactions, a special concern in the drug-abusing population, will be determined. The second seeks to evaluate further the most effective drug particles with the goal of quantitatively determining the subset of immunocytes in its tissue locale (gastrointestinal associated lymphoid tissue, bone marrow, lymph node and brain) that harbor residual virus. The, third will use combinations of antiretroviral and immune modulatory agents (for example, an indoleamine 2,3-dioxygenase inhibitor (IDIO, GSK3176181A) to induce HIV-1gag/pol-specific cytotoxic lymphocytes with the ?putative? goal of viral eradication. The most effective tested combinations for each of these aims will be cross-validated in SIV/SHIV infected rhesus macaques. The development of an injectable combination nanoART that can be dosed once every 6 months is realistic. Cabotegravir, is an already long-acting integrase inhibitor, currently in phase II trials. With a demonstrated half-life of 21?50 days following a single parenteral dose, it has the potential for an every three-month dose. Our own preliminary data offered in this project shows an up to 5-fold improvement in PK from what is being administered to eople with an even larger increase in drug reservoir targeting. This combined with the generation of polymer-encased pro-drugs of lamivudine and abacavir with markedly extended drug half-lives makes combination long-acting therapy realistic and without our grasp. On balance, we also acknowledge the obstacles. But balanced prior scientific successes, broad in-house technical experience and resources including trained personnel, equipment and infrastructure can meet the challenges. Further support includes access to developed and new antiretrovirals with support from GlaxoSmithKline and drug libraries. NanoART will include cell targeting to monocyte-macrophages and to subcellular organelles (late and recycling endosomes). We posit that modifications made of formulations in size, composition, coating, and charge will enhance nanoART cell uptake and potentiate subsequent drug delivery and release into viral reservoirs. These include susceptible CD4+ T lymphocytes. The establishment of the Nebraska Nanomedicine Production Plant, a good manufacturing production facility now housed at the medical center has enabled improved scale-up production of nanoART.